ABSTRACT Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer's disease (AD). Two-thirds of AD patients are apoE4 carriers, and apoE4 increases the risk and decreases the age of onset. Understanding how apoE4 causes neurodegeneration is critically important because it will guide the development of therapies to slow or possibly prevent apoE4-associated neuropathology. This proposal builds on our hypothesis that apoE4-associated neuropathology is related to the susceptibility of apoE4 to neuron-specific proteolysis. That proteolysis generates neurotoxic fragments that escape the secretory pathway and enter the cytosol where they interact with mitochondria, causing mitochondrial dysfunction and neurodegeneration. Human apoE4 carriers, even 20?30 years old, cognitively normal subjects, display brain glucose hypometabolism and impaired mitochondrial enzyme activity. In cultured neurons and transgenic mice expressing apoE4, we demonstrated low levels of mitochondrial respiratory enzymes and ATP production. Furthermore, we showed that apoE4-associated mitochondrial dysfunction can be reversed by apoE4 structure corrector molecules that convert apoE4 to the structurally and functionally more normal apoE3, thus preventing neurotoxic fragment generation, which interact with mitochondria and lead to neurodegeneration. The goal of this project is to establish how apoE4 and its fragments cause mitochondrial dysfunction and to define their mechanisms and pathways involved in neuropathology. We will examine how apoE4 alters mitochondrial metabolism, including ATP production and trafficking, and determine how the apoE4 structure correctors modulate mitochondrial activity. In addition, we will define how the apoE4 fragments interact with mitochondria and develop an assay to screen for small molecules that inhibit apoE4-mitochondrial interactions. Our studies identified a subset of mitochondrial proteins that are altered by apoE4 expression. We will couple the latest proteomic techniques, including mass spectroscopy and affinity purification, with CRISPR/Cas9- based validation to enhance our understanding of the pathways underlying apoE4-induced neurodegeneration. These insights will expand potential approaches for AD therapy.